An intrinsic control element for translational initiation in class 1 integrons

Abstract

Summary Integrons are genetic elements able to capture anti‐biotic resistance and other genes and to promote their transcription. Here, we have investigated integron‐dependent translation of an aminoglycoside 6′‐N‐acetyltransferase gene (aac(6′)‐Ib7) inserted at the attI1 site. N‐terminal sequencing revealed that translation of this gene was initiated at a GTG codon, which is not part of a plausible translation initiation region (TIR). A short open reading frame (called ORF‐11) overlapping the attI1 site was probed by site‐directed mutagenesis for its contribution to aac(6′)‐Ib7 translation. When ORF‐11 and its TIR were deleted en bloc, translational efficiency dropped by over 80%, as determined with an acetyltransferase– luciferase fusion product. Invalidation of the ATG start codon of ORF‐11 or its putative Shine–Dalgarno sequence resulted in a decrease of over 60%, whereas the decrease was much less pronounced when the amino acid sequence of the putative ORF‐11‐encoded peptide was altered or when the distance between ORF‐11 and aac(6′)‐Ib7 was doubled. This demonstrates that aac(6′)‐Ib7 translation is dependent upon the translation of ORF‐11, but almost certainly not upon the corresponding peptide. These results lead us to conclude that an intrinsic short ORF present in the 5′‐conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR‐deficient anti‐biotic resistance genes.

The Integron: Adaptation On Demand

“…In class I integrons, a small ORF (orf11) preceded by a functional RBS has been found in the attI1 site. This RBS is present in all transcripts generated from the P C promoters, and accounts for a significant part of the expression of cassettes devoid of an RBS (68).…”

The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes

“…These differences are likely to reflect different capacities to move to new sources of gene cassettes, but do they also reflect diversity in integron functions? It has been suggested that class 1 integrons have a greater capacity to express cassette-associated genes than other integron classes (Hanau-Bercot et al, 2002) and even within class 1 integrons different versions of P c are known, which vary significantly in their promoter strength Levesque et al, 1994). The issue of variation in integron function is particularly significant in the case of CIs since they can form long-term associations with specific bacterial lineages (Gillings et al, 2005; Rowe-Magnus et al, 2003, 2001).…”

Riboswitch control of induction of aminoglycoside resistance acetyl and adenyl-transferases

“…Expression of the resistance gene is delicately balanced; both of the aac/aad reporter constructs display elevated background levels of reporter gene expression in the absence of added aminoglycosides. Our current view is that lower levels of translation through the integron transcript and leader peptide as proposed for Citrobacter freundii 42 contribute to the background levels of reporter gene expression that we observe, and that the riboswitch acts as "dimmer switch" 67,68 in response to sub-lethal doses of the antibiotic to induce expression of resistance genes. It is intriguing to speculate as to whether the Citrobacter freundii RNA might also act as a riboswitch in response to added aminoglycosides.…”

“…39,41 Although the mechanism of insertion and transcription of integron DNA is becoming better understood, there have been fewer studies of the mechanisms of translation within integron sequences. 40,42 Hanau-Bercot et al studied the expression of the aac(6_ )-Ib7 gene cassette from Citrobacter freundii 42 and concluded that expression of aac was regulated at the level of translation of the leader peptide, and that the integron transcript functioned to allow initiation of translation at inserted gene cassettes that do not have correct ribosome binding sites 42 (reviewed in ref. 40).…”

“…The stop codon is located in the attI1 site but is created during gene capture and originates from the inserted gene cassette. 34,40,42 The downstream sequence by a complementary anti-SD sequence that blocks ribosome binding; antibiotic binding induces a structural transition that unmasks SD2 for ribosome binding leading to translation of the resistance gene (Fig. 1B).…”